Studies On Cell Wall Elongation Of The Stipe Cell Wall Of P. Cinerea Glucanase Recombinant Heat-killed Bacteria

The cell wall of fungi wraps around the protoplast.It not only provides sufficient mechanical strength for the cell against osmotic pressure and mechanical damages but also maintains the plasticity to allow the cell to grow,divide and expand.A variety of cell wall-associated hydrolases are likely to participate in the branch,cross-links of polysaccharides and the maintenance of cell wall plasticity in morphogenetic process.Previously,the model about hydrolases participating in stipe cell wall extension of basidiomycetes was proposed.It was that the breakage and subsequent reformation of cross-link bonds under the action of hydrolytic and transglycosylation activities of chitinases and glucanases made insertion of new cell wall components,chitin microfibril rearrangement and led to cell wall extension.But there is no experimental evidence to support the model.Thus,another opposite model was proposed that the stipe cell wall extension was due to the creep of polymers in cell wall caused by continuous breakage and reformation of hydrogen bond between the glucan chains and passive re-orientation of the transverse chitin chains.Experimental evidence about this model was also absent.This study attempts to extract endogenic proteins with elongation activity from Coprinopsis cinerea to reveal the molecular mechanism of stipe elongation.Firstly,the pilei just autolyzing of Coprinopsis cinerea are used to extract the protein with elongation activity.Crude extracts can reconstitute heated-inactivated stipe cell wall extension.After the crude extracts are subjected to salting out,dialysis,desalting on a P6 column,the active fraction is applied to a CM-Sepharose Fast Flow chromatography column and a fraction I-2 with the activity of glucanases and elongation activity is obtained.After the further separation and purification of fraction I-2,II-2-1 and II-2-2 with only glucanase activity but no activity of reconstituting heated-inactivated stipe cell wall extension are obtained and are identified as endo-1,3-?-glucanase(ENG)and extracellular ?-glycosidase(BGL2),respectively.The mixture of purified ENG and BGL2 reconstitutes heated-inactivated stipe cell wall extension.In addition,?-1,3-glycosidase(BGL1)is obtained.It is likely that two synergistically acted proteins separated by further isolation and purification result in loss of reconstituting stipe cell wall extension.We further investigate the characteristics and mechanism of glucanases synergistic reconstitution stipe elongation using recombinant ENG and BGL2.ENG(100 ?g/m L)and BGL2(1000 ?g/m L)don't reconstitute stipe cell wall extension alone,while the mixture with ENG(25 ?g/m L)and BGL2(260 ?g/m L)reconstitutes the heat-inactivated stipe elongation.The activity of stipe wall extension caused by protein synergistical action increases with the enzyme concentration increases and is affected by the p H of buffer.The maximum activity of synergistically reconstituting the heat-inactivated stipe elongation was at p H 3.0 and the activity decreases with the increase of p H value.And the p H of the maximum activity is not consistent with the optimal p H of ENG and BGL2,which is possibly on account of the easily broken hydrogen bonds between polysaccharide chains of cell wall in acid solution.The elongation activities of ENG and BGL2 synergistically reconstituting the heat-inactivated stipes from different region of 60 mm fruiting body show big differences.It is that the activity from the maximum to minimum is successively apical,medium and basal.At the same time,ENG and BGL2 synergistically reconstitute the heat-inactivated apical stipe elongation of Flammulina velutipes.Further,the products released from cell wall are analyzed.ENG alone releases glucose and three oligosaccharide products,and BGL2 only releases glucose.When stipe is incubated by the mixture of above two proteins,the oligosaccharide products released by ENG are degraded,with a large amount of glucose released.Based on above results,we hypothesize that ENG and BGL2 are likely to synergistically degrading the glucan to reconstitute heat-inactivated stipe cell wall extension.The hydrolysis toward alkaliinsoluble cell wall components shows that BGL2 mainly enhances the hydrolytic activity of ENG toward cell wall.In addition,high concentration of ENG(200 ?g/m L)alone can reconstitute cell wall extension and shows concentration-dependent characteristic,with releasing five products,among which four products are the same as that released by the low concentration of ENG.The BGL2 in mixture solution is replaced by ?-1,3-glycosidase(BGL1)or exo-1,3-?-glucanase(EXG)and is found that the mixture solution including ENG and BGL1/EXG reconstitutes wall extension and their extension curves are similar to that of ENG and BGL2.And the reconstituted cell wall extension activity increases with the enzyme concentration increases and is buffer p H-dependent.Similarly,the maximum reconstituted extension activity is at p H 3.0,which does not correspond to the optimum p H of the enzyme.In addition,we find that the mixture of EXG and BGL2 /BGL1doesn't reconstitute heat-inactivated apical stipe elongation.EXG or BGL1 at the high concentration alone don't reconstitute the heated-inactivated stipe wall extension,similar with BGL2.When ENG in mixture solution is replaced by endo-1,3(4)-?-glucanase(ENG16A)which also belongs to glycosyl hydrolase family 16,ENG16 A and BGL2/BGL1/EXG can synergistically reconstitute the heat-inactivated apical stipe elongation.By exploring the characteristics of ENG16 A and BGL2 synergistically reconstituting stipe wall extension,we find that the reconstituted wall extension activity is also enzyme concentration-dependent and p H-dependent,and the characteristic of p H-dependent is the same as that of ENG and BGL2.Identification of released products combined reveal the enzymatic hydrolysis mechanism of glucanases synergistically reconstituting heat-inactivated stipe wall extension,in which endo-1,3-?-glucanases play a main role,supplemented by other exo-glucananses and glycosidases synergistically degrading the cell wall skeleton structure.The exo-glucananses and glycosidases play a role in enhancing the activity of endo-1,3-?-glucanases.It is proposed that ?-1,3-glucan degrading enzymes are regulated by maintaining the low concentration ENG and regulating the expression levels of exo-glucanases and glycosidases,to maintain the cell wall plasticity and remodeling.According to the m RNA expression level analysis combined with western blot,the expression levels of these glucanases in apical region are highest than other regions in60 mm fruiting body and they are possibly associated with stipe cell wall extension in Coprinopsis cinerea and synergistically function in vivo,with the enzyme proportion used in synergistically reconstituting cell wall extension basically consistent with that of m RNA expression level.And the expression level of ENG16 A in apical region is5.79 times that of ENG,and the expression levels of ENG in different pileus(0°,90°,180°)is 4.84,211.8 and 363.6 times of ENG16 A,respectively.The difference in expression level between ENG and ENG16 A implies that ENG may mainly involve in the pileus expansion,while ENG16 A mainly involves in the elongation of the stipe.The ultrastructure of glucanases-reconstituted cell wall extension is observed by FESEM.It is found that the structure of the inner surface of the cell walls does not show fissure,but is different from that of the chitinases-reconstituted cell wall extension showing broken microfibrils.Moreover,glucanase-reconstituted wall extension initials at a high extension rate and extension rate gradually decreases to near zero until the end of experiment after about 60 min,while chitinase-reconstituted wall extension continues to extend at the initial extension rate until the end of the experiment.The stipe was not broken when high concentration of glucanase reconstituts cell wall extension,while the stipe is easy to be broken when high concentration of chitinase reconstituts cell wall extension.It is presumed that other polysaccharides such as ?-1,6-glucan,galactomannan,except for ?-1,3-glucan,may exist in cell wall of Coprinopsis cinerea,cross-linked with chitin,which makes glucanase-reconstituted wall extension different from chitinase-reconstituted wall extension.The mixture of glucanase and chitinase can reconstitute heated-inactivated cell wall extension,while the low concentration of glucanase or chitinase can't do it alone.The stipe elongates at the high rate at first and then is broken successively during the experiment,when the high concentration of glucanase and chitinase reconstitutes stipe wall extension.The results show glucanase and chitinase can synergistically reconstitute heated-inactivated stipe wall extension.The stipe acted by high concentration of the mixture glucanases and chitinases is broken easily,which reveals the balance of glucanases and chitinases ensure the stipe elongation.And the natural stipe wall acid-induced extension releases the glucose,N-acetylglucosamine and Nacetylglucosaminebiose.It is proposed that glucanases and chitinases participate in the elongation growth of Coprinopsis cinerea,and that the extension of the cell wall is caused of hydrolysis,relaxation and remodeling of chitin-glucan core structure.